Streptococcus pneumoniae is one of the leading causes of infectious morbidity and mortality in the world, responsible for a large spectrum of infections such as otitis media, pneumonia, bacteremia and meningitis {Hausdorff 2005, McCullers 2001}. The emergence of antibiotic-resistant strains of this micro-organism has further underlined the need for providing effective prophylactic vaccination {Lynch, III 2005, Bridy-Pappas 2005}.
Current vaccines are composed of epidemiologically dominant serotype-based selections of pneumococcal capsular polysaccharides, conjugated or not to a carrier protein {Dagan 2004, Fedson 2004, Mbelle 1999, Smart 1987}. However, the vaccine formulations do not cover all serotypes of this micro-organism, which might particularly be of relevance in certain regions of the globe with different dominant serotypes {Dagan 1992}. In addition, one may expect that the use of serotype-specific vaccines could allow at term the positive selection of non-vaccine serotypes {Nunes 2008, Singleton 2007}.
An alternative approach involves the development of vaccines that target common pneumococcal antigens. Among multiple candidates, the Pht protein family, restricted to the genus Streptococcus, comprises promising ones, being well-conserved across the pneumococcal species {Hamel 2004, Zhang 2001}, and being antibody targets in infected individuals and protective upon challenge in immunized mice {Beghetto 2006}. Originally, this protein family was independently reported by three groups, and three separate denominations were used: Pht (for pneumococcal histidine triad) {Adamou 2001}, Php (for pneumococcal histidine protein) {Zhang 2001}, and BVH {Hamel 2004}. Those proteins are characterized by a histidine triad motif, HxxHxH, repeated five to six times in their amino-acid sequences. Four members of this family have been described: PhtA (BVH-11-3), PhtB (PhpA/BVH-11) and PhtD (BVH-11-2) that share up to 81% sequence identity, and PhtE (BVH-3) that diverges from the three other proteins, showing only up to 35% identity with them. It is a longer protein, the only one with six repeats of the histidine triad motif. In mouse immunization studies, all members of the Pht family have been shown to afford a high level of protection to subsequent pneumococcal infection with a number of different strains/serotypes {Adamou 2001, Hamel 2004, Ogunniyi 2007, Wizemann 2001, Zhang 2001}.
Despite their potential importance in vaccination against S. pneumoniae, the biological function of these proteins has yet to be determined. Results from antibody-labeling and flow cytometry experiments demonstrated that the Pht proteins are exposed on the surface of the encapsulated bacterium {Hamel 2004}, which is in agreement with their relevance as vaccine target. By signature-tagged mutagenesis, it has been suggested that PhtA, PhtB, and PhtD are involved in lung-specific virulence {Hava 2002}, without further indication about their biological function. Among their putative roles, neutralization of the complement factor C3b has been suggested {Hostetter 1999, Ogunniyi 2009}, which implies that they would interfere with phagocytosis. Besides that, a role in adherence is also suspected. Indeed, a genetic link between phtD and lmb, the latter encoding a putative laminin adhesion protein {Spellerberg 1999}, has been reported {Panina 2003}. At last, due to the high number of histidine residues in the histidine triads, it has been suggested that the Pht proteins may be involved in DNA and/or metal binding {Adamou 2001}. More specifically, some studies highlighted a link between the Pht family and zinc. Indeed, AdcR-binding sites have been found in the upstream regions of the pht genes, AdcR being described as a transcription factor that regulates zinc uptake {Panina 2003}. Furthermore, the crystal structure of a portion of PhtA revealed the presence of zinc ions bound to a histidine triad domain {Riboldi-Tunnicliffe 2005}. It is not clear, however, whether zinc scavenging or transport is the function of those proteins, or whether zinc rather plays a conformational or functional role.
Important aspects that need to be addressed for vaccine candidates are their level of expression and associated regulation, their occurrence as well as their sequence variability. Therefore, we have addressed these different aspects with regard to the Pht proteins.
Streptococcus pneumoniae elicits different disease states exhibiting different pathologies depending on the site at which the pneumococcal population expands. Septicaemia occurs where the S. pneumoniae enters to blood steam, whereas pneumonia occurs where S. pneumoniae multiplies in the lung. S. pneumoniae is also an important pathogen in otitis media infections. S. pneumoniae can also enter the cerebrospinal fluid to cause meningitis.
There is a need to develop better pneumococcal vaccines which are able to target specific pneumococcal diseases and provide optimal protection against a particular form of pneumococcal disease.
Accordingly there is provided a method of treating or preventing Streptococcus pneumoniae infection wherein the Streptococcus pneumoniae infection occurs in an environment where the concentration of Zn2+ and/or Mn2+ is sufficiently low to upregulate the expression of at least one PhtX protein in the Streptococcus pneumoniae; comprising the step of administering a pharmaceutically effective amount of the PhtX protein to a human patient.
In a second aspect of the invention there is provided an immunogenic composition comprising a pharmaceutically effective amount of an isolated PhtX protein for use in the treatment or prevention of a Streptococcus pneumoniae infection wherein the Streptococcus pneumoniae infection occurs in a human patient in an environment where the concentration of Zn2+ and/or Mn2+ is sufficiently low to upregulate the expression of at least one PhtX protein in the Streptococcus pneumoniae. 
In a third aspect of the invention there is provided a use of a pharmaceutically effective amount of an isolated PhtX protein in the manufacture of a medicament for the treatment or prevention of a Streptococcus pneumoniae infection wherein the Streptococcus pneumoniae infection occurs in a human patient in an environment where the concentration of Zn2+ and/or Mn2+ is sufficiently low to upregulate the expression of at least one PhtX protein in the Streptococcus pneumoniae. 